LC-ESI-HRMS-Based Evaluation of Antioxidants as Additives to Inhibit Genotoxic Nitrosamine Formation in Metformin Hydrochloride Tablets

Abstract

Nitrosamines, particularly N-nitrosodimethylamine (NDMA), have raised significant regulatory and safety concerns due to it's genotoxic and carcinogenic properties. Although NDMA formation in metformin hydrochloride (MET) tablets under nitrosating conditions is well-established, the potential of antioxidants to inhibit this impurity remains underexplored. This study evaluated the effectiveness of antioxidant additives, trolox (TRX), ascorbic acid (ASA), and alpha-tocopherol (Alpha- T), in minimizing NDMA formation during storage. MET tablets were formulated into three groups: (a) spiked with sodium nitrite (NaNO₂), (b) spiked with NaNO₂ and antioxidants, and (c) containing antioxidants without NaNO₂. A total of 23 batches were manufactured and subjected to both accelerated and long-term stability conditions for three months. NDMA levels, quantified by validated LC-HRMS method, showed significant increase under stress conditions, which were attenuated by antioxidant inclusion. TRX (5%) demonstrated the highest inhibition of NDMA formation (62.16%) under accelerated storage (Day 90), followed by ASA (31.80%) and Alpha-T (32.28%). Similar trends were observed in long term conditions. In vitro Caco-2 permeability assay revealed a 2.17-fold increase in MET transport with 10% TRX. Genotoxicity studies using the in vitro comet assay in HepG2 cells confirmed TRX as the most effective antioxidant in reducing NDMA-induced DNA damage. These results demonstrate that antioxidant incorporation can effectively control NDMA formation and improve bioavailability in MET tablets, offering a formulation-based approach to mitigate nitrosamine risk.

Keywords: NDMA; antioxidants; bioavailability; comet assay; metformin hydrochloride.